The miRNeasy Mini Kit enables purification of total RNA, which includes RNA from approximately 18 nucleotides (nt) upwards from all types of animal tissues and cells, including difficult-to-lyse tissues. Alternatively, an miRNA-enriched fraction and a total RNA (>200 nt) fraction can be purified separately (for separate purification, an RNeasy MinElute Cleanup Kit is required). The miRNeasy Mini Kit is used for low-throughput RNA purification using spin columns. Copurification of miRNA and total RNA using the miRNeasy Mini Kit can be automated on the QIAcube.

The miRNeasy Mini Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Effective purification from a range of starting amounts.

Total RNA was purified from [A] 102–107 Jurkat cells using the miRNeasy 96 Kit or [B] a dilution series of rat lung tissue homogenate from 20 mg to 200 ng using the miRNeasy Mini Kit. miRNA-enriched fractions (<200 nt) were also isolated from the same samples. Purified RNA was used as a template in quantitative, real-time RT-PCR assays for the miRNA miR-16.

Efficient copurification from a wide range of tissues.

Total RNA including miRNA was purified from 25 mg of a range of RNAlater stabilized rat tissues using the miRNeasy 96 Kit. Purified RNA was used as a template in quantitative, real-time RT-PCR assays for the miRNA miR-16 and for the larger mRNA of the PGK1 gene. Results showed successful detection of both PGK1 mRNA (large RNA) and miR-16 (small RNA) from the same eluates.

miRNeasy Mini Kit outperforms TRIzol.

Total RNA including miRNA was purified from a range of amounts of Jurkat cells using the miRNeasy Mini Kit or TRIzol Reagent. Purified RNA was used as a template in quantitative, real-time RT-PCR assays for the miRNA miR-16. Results showed that CT values were lower after purification using the miRNeasy Kit, indicating that higher amounts of miRNA were purified than using TRIzol. miRNA was effectively purified from as little as 1 x 102 cells using the miRNeasy Kit. In contrast, no miRNA was detected after 40 PCR cycles from 1 x 102 cells when TRIzol was used for purification.

Effective enrichment of small RNA.

miRNA-enriched fractions were purified from 3 x 106 HeLa cells using either the miRNeasy Mini Kit or a kit from Supplier AIV. Equal RNA amounts from miRNA-enriched fractions were run on a 1.5% gel. For comparison, 20mer and 21mer synthetic RNAs were also run on the gel. The miRNA-enriched fraction prepared using the miRNeasy Kit showed a smear of RNAs of different sizes down to approximately 18 nt. In contrast, in the miRNA-enriched fraction prepared using the kit from Supplier AIV, primarily tRNA and 5S rRNA were visible.

Highly pure RNA without phenol carryover.

Total RNA including miRNA was purified from 1 x 106 Jurkat cells using the miRNeasy Mini Kit or TRIzol Reagent. The absorbance spectra showed the OD maximum for RNA purified using the miRNeasy Mini Kit was at 260 nm. In contrast, the OD maximum was greater than 260 nm when TRIzol was used for purification, indicating phenol carryover. In addition, the OD230 measurement was higher in the TRIzol-prepared RNA, indicating salt carryover from the TRIzol Reagent.

miRNeasy procedures.

The miRNeasy Mini Kit enables purification of total RNA which includes RNA from 18 nucleotides (nt) upwards. Alternatively, an miRNA-enriched fraction and a total RNA (>200 nt) fraction can be can be purified separately. A separate miRNA-enriched fraction may be required to reduce background in particularly sensitive downstream applications. Separate purification procedures using the miRNeasy Mini Kit require additionally the RNeasy MinElute Cleanup Kit. For purification in a 96-well format, we recommend the miRNeasy 96 Kit.

Performance

Purification of total RNA including miRNA with the miRNeasy Mini Kit allows direct comparison of miRNA expression levels with those of housekeeping reference genes or any other mRNA of interest. Alternatively, for some sensitive downstream applications, an miRNA-enriched fraction without larger RNA may be required. miRNeasy Kits enable efficient enrichment of RNA down to approximately 18 nt in size. The presence of very small RNAs is clearly visible after purification using an miRNeasy Mini Kit, in contrast to samples prepared using an alternative kit (see figure "Effective enrichment of small RNA"). miRNeasy Kits efficiently purify RNA from both tissues and cells, even when low amounts of starting material are used. When RNA was purified from decreasing amounts of samples, CT values increased linearly, indicating effective miRNA purification from a wide range of starting material (see figure "Effective purification from a range of starting amounts").

RNA can be purified with the kit from a variety of tissues and cells, including difficult-to-lyse tissues (see figure "Efficient copurification from a wide range of tissues"). miRNeasy procedures eliminate the possibility of contamination with salts or phenol which could interfere with later analyses (see figure "Highly pure RNA without phenol carryover"). In addition to higher purity, miRNeasy Kits offer superior yields to alternative methods of miRNA purification, such as using TRIzol Reagent (see figure "miRNeasy Kit outperforms TRIzol"). RNA prepared using the miRNeasy Mini Kit is highly pure and ready for use in sensitive downstream applications.

Principle

The miRNeasy Mini Kit combines phenol/guanidine-based lysis of samples with silica-membrane–based purification of total RNA. QIAzol Lysis Reagent, included in the kit, is a monophasic solution of phenol and guanidine thiocyanate, designed to facilitate lysis of tissues, to inhibit RNases, and also to remove most of the cellular DNA and proteins from the lysate by organic extraction.

Procedure

The miRNeasy Mini Kit enables purification of total RNA which includes RNA from 18 nucleotides (nt) upwards. Alternatively, an miRNA-enriched fraction and a total RNA (>200 nt) fraction can be can be purified separately (see flowchart "miRNeasy procedures"). The separate miRNA-enriched fraction may be required to reduce background in particularly sensitive downstream applications. For this specialized protocol, enabling enrichment of miRNAs and other small RNAs (<200 nt), the RNeasy MinElute Cleanup Kit is required.

Purification of total RNA including miRNA with the miRNeasy Mini Kit can be automated on the QIAcube. The innovative QIAcube uses advanced technology to process QIAGEN spin columns, enabling seamless integration of automated, low-throughput sample prep into your laboratory workflow. Sample preparation using the QIAcube follows the same steps as the manual procedure (i.e., lyse, bind, wash, and elute) enabling you to continue using the miRNeasy Mini Kit for purification of high-quality RNA. More information about the automated procedure is available from the

relevant protocol sheet.

Applications

The miRNeasy Mini Kit allows purification of miRNA with total RNA for use in a variety of applications, including: